This project is concerned with the elucidation of the mechanism of the mammalian heart mitochondrial and Rhodospirillum rubrum energy- linked pyridine dinucleotide transhydrogenase reaction: NADH plus NADP ion plus X approximately Y yields (reversibly) NAD ion plus NADPH plus X plus Y where X approximately Y is a high-energy intermediate or state of the oxidative phosphorylation system. Problems to be investigated with the bovine heart enzymes are (a) localization of the transhydrogenase in the inner mitochondrial membrane (b) stoichiometry of proton translocation coupled to transhydrogenation in submitochondrial particles (c) pyridine dinucleotide and energy dependent conformational alterations of the enzyme (d) demonstration of a possible reduced enzyme intermediate (e) elucidation of amino acid residues of the enzyme involved in proton translocation (f) purification of bovine heart transhydrogenase and reconstitution of energy-linked transhydrogenation by incorporation of the purified enzyme into proteoliposomes. Further studies on the R. rubrum transhydrogenase include (a) purification of both the soluble component and the membrane component (b) determination of the specificity and locale of the substrate binding sites on the two enzyme components (c) determination of the component containing a reduced enzyme intermediate and (d) further elucidation of NADP(H) induced conformational changes of the membrane bound component.